Journal of the Pancreas Open Access

Keywords

Apigenin; HMGB1; Interleukin-6; Interleukin-18; Pancreatitis;Resistin

Abbreviations

ACP Acute Pancreatitis; APApigenin; Ap groupApigenin group; C group Control group; Damage Associated Molecular PatternDAMP; HMGB1High Mobiity Group Box 1; IL-6Intereukin-6; IL-18Intereukin-18;Janus Kinase/Signal Transducer and Activator JAK/STAT;RResistin; S groupSham group; TCM Traditional Chinese Medicine; TLRs Toll-Like Receptors

Introduction

ACP is characterized by inflammation of the pancreas with involvement of neighboring tissues or distant organs/systems. In mild form, there is recovery without complications. In severe form, there are local and systemic complications such as renal and pulmonary failure as well as bowel related complications [1,2]. Although the exact mechanisms that initiate an episode of ACP are not clearly understood, all cases share the same inflammatory and repair pathways. Unregulated activation of trypsin within pancreatic acinar cells causes local inflammation and leukocyte turnout at the peripancreatic vascular network.

Within a few hours activated monocytes are attracted, further stimulating local and systemic hyperproduction of pro-inflammatory cytokines and interleukins such as IL-6 and IL-18 [3,4]. HMGB1 and CD45 play an important role in mediating inflammation and regulating immune responses, respectively. HMGB1 acts as pro-inflammatory cytokine when released extracellularly under stress conditions, like ACP [5,6]. CD45 is present on pancreatic acinar cells having various roles in activation-induced signaling in leukocytes, related to the inflammatory responses [7,8]. Moreover, according to recent studies, a new effective indicator that can predict the severity of ACP and is used for its assessment is R [9]. Studies in early stages of ACP have difficulties in being performed on humans, as the diagnosis is established when pancreatic injury has already been initiated. As a consequence there is missing information about the facts that occur during the initial stages of ACP. This gap can be filled through research on experimental animal models of ACPin order to enrich our knowledge [10]. The beneficial role of AP, a flavonoid substance, has been described in several studies [11]. It is proven that has anti-inflammatory, anti-oxidative, anti-carcinogenic, anti-mutant, antiallergic and anti-osteoporotic effects [12]. The aim of this study is to determine whether AP has positive effect on experimentalmodel of ACP. In order to accomplish it, we determined IL-6, IL-18 and R levels in serum and HMGB1 and CD45 expression in intestinal tissue of animal models with ACP after AP administration at different time points.

Materials and Methods

Animals and Study Design

126albine male wistar rats, 90-120 days old, with a median weight of 280g (IQR=220-350g), were used. The animals were maintained in groups of four, in polycarbonate cages, under controlled conditions of temperature (22oC – 25oC), humidity (55%-58%) and light (12/12 hours light/dark) with free access to rat food and tap water ad libitum.

All animals were supplied by Pasteur Institute, Athens, Greece. The experiments took place at the approved Experimental Research Center of ELPEN Pharmaceuticals, Athens, Greece, and the immunohistochemistry and ELISA assays took place at the laboratories of Medical School of Democritus University of Thrace. All experimental procedures conformed to the principles laid by the National Research Council Guide for the Care and Use of Laboratory Animals and the Directive 86/609 of the European Union (protocol number K/2284).

The experimental animals were randomly assigned into 3 groups: 1) S group (S; n=20), in which animals underwent midline laparotomy without induction of ACP or AP administration;2) C group (C; n=56), in which animals underwent midline laparotomy with induction of ACPvia ligation of the common bilio-pancreatic duct; and 3) Ap group (Ap; n=50), in which animals underwent midline laparotomy with induction of ACP, followed by postoperative per osadministration of AP. The animals of each group were further subdivided into five subgroups according to the euthanasia time, i.e., 6h (C6, Ap6),12h (C12, Ap12),24 h (C24, Ap24),48h (C48, Ap48) and 72h (C72, Ap72). In the Apgroup, the substance was administered as follows: i) Ap6 (n=10): 1 dose of 4mL, ii) Ap12 (n=10): 1 dose of 4mL,iii) Ap24 (n=10): 1 dose of 4mL, iv) Ap48 (n=10): 2 doses of 4mL with a 24 h interval, v) Ap72 (n=10): 3 doses of 4mL with 24 h intervals, followed by euthanasia. The overall study design was based on a previous publication by members of our research group [13].

Protocol of Experimentally Induced ACPin Rats

Induction in anaesthesia was performed by placing each experimental animal in a special glass box connected to isoflurane supply for about 2 to 3 min, followed by subcutaneously injected 0.25mL of Butorphenol 10 mg/mL (Dolorex;Intervet/Schering/Plough Animal Health, Boxmeer, Holland). Endotracheal intubation was performed under direct laryngoscopy by trained staff using a 16-G venous catheter connected to an Ugo-Basile ventilator for rodents (Harvard Apparatus, Holliston, MA), which was adjusted at tidal volume of 3 mLand respiratory rate of 70 breaths/min. Chest expansion and lung auscultation was indicating a proper intubation.

With the experimental animal placed in a supine position, a midline laparotomy of about 3cm was performed under sterile conditions. After the entry in the abdominal cavity the bilio-pancreatic duct was identified and ligated close to the duodenum with a non-absorbable 4/0 silk suture for the induction of ACP [14]. 1 mL of NaCl 0.9% was instilled in the abdominal cavity before closing the lineaalba with vicryl 3/0 suture and the skin with nylon 3/0 suture. Postoperatively the animal was fed ad libitum while analgesia (2mL ofbutorphenol, Dolorex) was administered subcutaneously every 4 hours, and then, according to the animal’s needs based on their clinical picture. In S group, operation was followed by identification and manual mobilization of the pancreas without ligation of the bilio-pancreatic duct or further action before the abdominal closure. In Apgroup, after the surgical ligation of the duct, once the animal was becoming conscious, 4 mL of prepared AP solution was administered orally.

Nine rats died before scheduled euthanasia, two during the surgical experiment due to intubation complications from the Cgroup and seven post-surgery, one from the Apgroup and six from the Cgroup. These animals were not included in the statistical analysis.

AP Solution Preparation

AP was suppliedby Sigma-Aldrich, Taufkirchen, Germany in vials with >99% purity by thin layer chromatography, as yellow powder with tan cast. AP was dissolved as follows: 660 mg AP was dissolved in 500mL corn oil, 2mL dimethyl sulfoxide and 10mL Tween 80. The final concentration was 5 mg AP in 4mL solution.

Immunohistochemistry

The immunochemistry procedure was performed in paraffin-embedded tissue section by using the biotin / streptavidin technique. The kits that were used were Super Sensitive Onestep Polymer HRP detection system (QD 630XAKE, Biogenex) for HMGB1 and CD45. The antibody used for the detection of HMGB1 was the HMGB1 polyclonal antibody in dilution 1:250 and incubation time 60min (rabbit polyclonal, PA185-16926, Thermo Scientific) and forCD45 was the monoclonal antibody in dilution 1:250 and incubation time 60min (mouse monoclonal, sc53047, Santa Cruz). A scoring system for all antibodies was assigned according to the proportion of cells with cytoplasmic staining as follows:i) 0: negative (≤10% of stained cells); ii) 1:low (>10% and ≤30% of cells stained); iii) 2: moderate (>30% and ≤70% of cells stained) and iv)3: high (>70% cells stained).

Determination of Serum IL-6,IL-18 and Resistin

IL-6, IL-18and resistinprotein levels were determinated in serum samples by ELISA according to the manufacturer’s instructions (IL-6: rat IL-6, ER 2116, Thermo Scientific, USA;IL-18: rat IL-18, orb 50132, biorbyt, UK;resistin: E02R0351, amsbio, UK). Protein levels were interpolated from the corresponding standard reference calibration curves. BCA protein assay (Thermo Scientific, USA) was used for total protein determination.

Statistical Analysis

The continuous variables are demonstrated as median with range (min-max). The non-parametric tests Mann- Whitney and Kruscal-Wallis were used to compare the medians of continuous variables in different categories of qualitative variables (group and time). In the case of a statistically significant result of Kruscal-Wallis test, Dunn’s post hoc tests were carried out on each pair of groups. The Bonferroni adjustment was used. As post-hoc tests are more conservative than the global test there was a case where the results of Kruscal-Wallis were statistically significant but pairwise comparisons were not. Moreover, if the number of observations in each group or time point was less than four, a sensitivity analysis was conducted keeping only the groups and time points with more than four observations. As the study variables were classified into multiple categories, the non-parametric tests were used due to the small samples. Relationships with a P-value (p) ≤ 0.05 were considered as statistically significant. All reported P-values are two-sided. The data was analyzed in using the Statistical Package for the Social Sciences 25.0 (SPSS Inc., Chicago, IL, USA).

Results

AP ReducesIL-6in Rats with Experimental ACP at 12h

IL-6 concentrations were lower in S group compared to C andAp groups, at every time point (Figure 1). More specifically, in S group, the initial value was the highest (mean: 61.10; range: 30.80 – 64.70) and diminuated gradually for every time point that followed (Figure 1).In C group IL-6 reached its highest value at 12h (mean: 129.30;range: 63.70 – 263.50) due to the inflammation caused by ligation of the bilio-pancreatic duct. At this time point of 12h, values in C and Ap groups almost coincided and presented their highest levels (mean: 162.50; range: 110.90 – 311.60). It is shown that after 12h, in the Apgroup the level of IL6 is reduced, leading to a steady decline, whereas in the C group, fluctuations of the level of IL6 are seen.Probably, the beginning of the anti-inflammatory effect of the substance starts at 12h, result which also reaches statistical significance from the pairwise comparisons between S and Ap groupsat 12h (p=0.007) and at 24h(p=0.005) (Table 1). From comparisons between the 5 time points for each group, in the Ap group, every time point combination presented statistical significant results (p<0.001), underlying the effect of APadministration (Table 2).

Table 1. Between group differences over the 5 time points.

Table 2: Within group differences over the time points.

AP Reduces IL-18 in Rats with Experimental ACP at 6 h

IL-18 remained at low levels in the S group, with a peak at 24 h time point (Figure 2). In the C group, it reached the highest value at 6 h time point, diminished until 12 h and then augmented until 48 h, where it started to fall again. In the Ap group, the onset value was the highest of all 3 groups (mean:2.03; range: 0.22-3.68) (Table 2). The effect of APstarted at 12h, followed by a huge drop in value of IL- 18 until 24 h, when it rose again. Another drop appeared at 48h with the lowest value at 72h. The only statistically significant result was at the onset point (p=0.028), in which Ap group showed the highest and the S group, one of the lowest values (Table 1).

AP Reduces HMGB1 Expression

HMGB1 expressions among the 3 groups reached statistically significant differences almost at all-time points (Table 1). HMGB1 presented low expressions in the S group, whereas in the C group, it increasedafter 12 h and reached its highest value at 24h (Table 2, Figure 3). The beneficial role of APstarted at 48h (p=0.02), while HMGB1 gained its lowest value in theAp group at 72 h time point (p=0.001) (Table 1), with statistical significance in pairwise comparison of 6h vs. 72 h (p<0.001) (Table 2).

AP PresentsAnti-Inflammatory Effect, as CD45Expression is Decreased

In the S group, CD45 remained low and stable at every time point (Figure 2). In the C group, it increased reaching its highest value at 72 h. On the contrary, in the Ap group, the expressions of CD45 coincidedwith those of the S group after 24h. Statistical significance for pairwise comparisons between the S and C groupswas found for every time point after 24h (24h: p=0.043; 48h: p=0.002; 72h: p<0.001) and between C and Ap at 48h (p=0.002) and 72 h (p<0.001) (Table 1). From the comparisons between the 5 time points for each group, both C and Ap groups presented statistical significant results for 6h vs. 72h (p=0.001) and 12h vs. 72h (p=0.011), underlying for the C group the upward trend of values and for the Ap group the downward trend of values(Table 2, Figure 4).

AP ImprovesInflammatory Effect, as Levels of RRemain at Low Points

Ιn both S and C groups, Rranged from low to high values without any specific pattern (Figure 5). On the contrary, the Ap groups presented the lowest values compared to C groups, with statistical significance at 12 h, 48h and 72h (p=0.002).

Discussion

ACP is an inflammatory process of the pancreas with adverse outcome. It is characterized by damage of peripancreatic tissues and/or distant organs, and it seems to rather comprise a variety of diseases than a sole one [15]. Pathophysiological mechanisms involved in the cause of ACP, especially these of the early stages of the disease, are under study. All mechanisms lead to release of cytokines and free radicals of oxygen, increasing the inflammatory process, the apoptosis and the oxidative stress [16].

Due to the continuous research interest on pancreatitis, severalexperimental models of pancreatitis have been developed. In our study, we used the experimental model of ligation of the bilio-pancreatic duct [14] which mimics acute obstruction similar to gallstone pancreatitis in humans.

Our study refers to the effect of a flavonoid, AP, on known inflammation indices such as IL-6 [3], IL-18 [4], HMGB1 [17], CD45 [18] and R [9] in rat serum and intestinal tissue. Flavonoids are phenolic phytochemicals, naturally existing in plants and abundant in particular plant species, vegetables and fruits. They are considered important constituents of the human diet, although their daily intake varies with dietary habits. The beneficial role of flavonoids is well known in the traditional chinese medicine (TCM). TCM is one of the most popular complementary and alternative medicine modalities worldwide for the treatment of ACP, and one of its most powerful medicines is scutellarin. In the last decade, further studies enhance scutellarin’s beneficial effect on inflammation, as a family member of flavonoids [19,20].Several properties have been ascribed to flavonoids including AP. It seems that AP downregulated Tumor Necrosis Factor–α in an experimental model of ACP, so it is expected, to also down-regulate other inflammatory cytokines [21]. AP is characterized by anticancerous, anti-oxidative, anti-inflammatory and other actions [12].

Ιn our study,AP, showed its anti-inflammatory effect, mainly at 12 h reducing IL-6 serum levels. Concerning IL-6, it acts via gp130 protein, leading the activation of Janus kinase/signal transducer and activator (JAK/STAT) signaling pathway [22,23]. In vivo studies proved that patients suffering from pancreatitis developed high serum values of IL-6 compared to healthy individuals (52).A recent study of Mrazeket al.which is in accordance with our findings showed that AP reduces IL-6 levels [11].

IL-18, is a member of IL-1 family cytokines. There are many studies that prove the presence of IL-18 in the blood of patients suffering from ACP. Moreover its values increase during mild and severe forms of the disease compared to healthy people [4]. In experimental models of ACP, high levels of IL-18 have also been described [24]. In our study, APshows its anti-inflammatory action between 12h and 24h, as we observe an important reduction of IL-18 value in Ap group. In general, IL-18 seems to be present at the early stages of ACP acting as immunomodulator of the inflammatory response especially in severe forms of the disease, however, without knowing the exact mechanisms of pathogenesis [24].

HMGB1 is a nuclear protein which regulates transcriptional process and genetic expression, while it plays an important rolein replication and repair of DNA. Loss of HMGB1 leads to structural and functional anomalies of the nucleus. Under stress conditions it is moved from nucleus to cytoplasm, where it mediates autophagy. Beyond its intracellular action, it can acts as extracellular protein as well, and more specifically as damage associated molecular pattern(DAMP) molecule. These molecules act as cytokines which through HMGB1 receptors or toll-like receptors (TLRs), activate specific signaling pathways mediating the inflammatory and immune response. According to experimental studies, oxidative stress is responsible for the translocation, release and action of HMGB1 [25,26].

Studies try to explain the relationship between HMGB1 and ACP. Most of them showed linear correlation of HMGB1 and other inflammatory parameters [25,27], while inone study, a reduction of HMGB1 levels was described through the action of siRNA that inhibited NFkB, decreasing the inflammation in severe ACP [28]. In general, HMGB1 expression in patients with ACP, seems to increase. Two are the possible mechanisms responsible for this. The first one is “HMGB1 circulation” [29], which is a phenomenon under study. According to this, reduction of HMGB1 through administration of anti-HMGB1 antibody causes reduction of the severity of ACP and vice versa. The second mechanismis based to the fact that in ACP, HMGB1 is released from the injured pancreas as well as from lungs and intestine that are also affected [30]. The results of our study are in total agreement with the majority of the studies, as in the C group we observed statistically significant increase of HMGB1 expression with time while in the Ap group we detected the positive effect of AP after 48 hours of the experiment. Moreover, at every time of the experiment there was statistically significant difference between two of the groups, undoubtedly pointing at the beneficial role of the substance’s administration on the intestinal mucosa [31].

CD45 is another antigen related with inflammation. As it was expected, in S group, CD45 remained low and stable for every time point. On the other hand, in both groups with ACP it increased, coming in line with the existing bibliography [32,33]. The AP started its action at 24hours, by down-regulating the expression ofCD45. To the rest of our knowledge, this is the first study that has noticed a great and undoubtableimpact of AP on CD45 of the intestine mucosa.

Concerning R, the lower levels of its concentration may correlate with the early action of AP. Generally, R is involved in the early stages of inflammation. Daniel et al.observed that R serum concentration in patients with ACP was significantly increased on the first, third and fifth day of hospitalization compared to the control group [34] while Xueet al. indicated in their study thatRmay be associated with the occurrence and development of AP and as a resultthe protein may be a valuable indicator for assessing the severity of ACP [35]. In our studythe beneficial role of APon Rwas also obvious.

As it is well known,ACP is also associated with acute lung and kidney injury.Recent studies have proven that the administration of anti-inflammatory substances, such aseugenoland AP, can protect kidneys and lung injury in an experimental model of ACP in rats [36,37]. In our study, we observedthe beneficial role of AP not only in serum parameters, but also in the intestinal mucosa. It seems that the therapeutic target of ACP is not just the pancreas but also other organs – targets of the disease, such as the intestine, the kidneys, the lungs.The prevention of multiple organ disfunction can be useful in diminishing the morbidity and mortality of the disease.

In the present study 4 mL of AP solution were administered every 24 hours containing 5mg of AP,as described previously. This dose proved to be capable to satisfy our hypothesis that AP is a potent anti-inflammatory agent [13], after ACP. The selection of the dosage was based on previous studies investigating daily dietary exposure of AP to humans [38]. Dosages of 100-200mg/kg or more induce cytotoxicity and produce phenoxyl radicals and reactive oxygen species [39,40].

Conclusion

In conclusion, AP administration in a rat experimental model appears to be beneficial on the examined parameters as it reduces theinflammation, caused in that experimental model ofACP. APadministration seems to be a promising therapeutic non-toxic agent which should be further tested in clinical use.

Acknowledgements

This study was funded with a scholarship by the Experimental, Educational and Research Center of ELPEN Pharmaceuticals (EERCE), Athens, Greece, which also provided the research facilities for this project.

Declerations of Conflicting Interest

The authors declare that there are no conflicts of interest.

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